Summary of Work: In preliminary studies reported previously the HIV viral coat protein, gp120, inhibited voltage-dependent calcium channels in a rat pituitary tumor cell line (GH4C1). We are investigating the possibility that gp120 acts through a G-protein coupled chemokine receptor to stimulate protein phosphatase activity, as we have demonstrated for other G-protein coupled receptors in GH cells. To obtain a defined and reliable source of gp120, we subcloned a secretion-directed cDNA sequence encoding the HIV coat protein, GP120 strin MN, with mammalian codons (Haas et al., 1996 Current Biology 6:315) into the mammalian exression vector, PCI-Neo (Promega Biotech) and transfected it into HelaS3 cells, a cervical carcinoma line. A subclone of HelaS3 with high stable GP120 expression was isolated, and the protein was purified from the conditioned medium. GP120 was purified by a single step purificationon a GP120 antibody affinity column by elution with high magnesium. We are testing the effects of this recombinant protein on ion channel activity in GH4C1 cells.